CN112997802A

CN112997802A - Cryopreservation method of wood rot fungi

Info

Publication number: CN112997802A
Application number: CN202110216801.1A
Authority: CN
Inventors: 张振宇; 孙姣; 李洁
Original assignee: Shandong Xiangyu Seed Technology Co ltd
Current assignee: Shandong Xiangyu Seed Technology Co ltd
Priority date: 2021-02-26
Filing date: 2021-02-26
Publication date: 2021-06-22

Abstract

The invention discloses a freezing preservation method of wood-rotting fungi, and relates to the technical field of edible fungi preservation. Which comprises the following steps: and (3) taking the wood chip particles as carriers, and after the hypha of the wood rot fungi to be preserved spreads and grows to the interior of the wood chip particles, performing freeze preservation on the wood chip particles growing the wood rot fungi to be preserved. The method provided by the invention has the following beneficial effects: the survival rate of the mycelium is high, the germination speed is high, the mycelium grows vigorously, and the problems that wood rot fungi are easy to pollute, mutate and even die in the using and passage processes, so that the strains are declined and the strains are lost are solved.

Description

Cryopreservation method of wood rot fungi

Technical Field

The invention relates to the technical field of edible fungi preservation, in particular to a freezing preservation method of wood rot fungi.

Background

The edible fungi can be divided into straw rotting fungi and wood rotting fungi according to the raw materials required by the cultivation of the edible fungi. The main raw materials for cultivating wood rot fungi are wood chips and cottonseed hulls of broad-leaved trees. So far, about 500 wood rot fungi are known in China. The wood rotting fungus is easy to pollute, mutate and even die in the using and passage processes, so that the strain is degenerated and even good strains are lost, and therefore the strain preservation is needed, the strain can keep good biological activity, does not degenerate and mutate, and does not pollute the strain due to regular transfer. The preserved strain needs to be as close to the original strain as possible to meet the requirements of research and use.

At present, the preservation of wood rot fungi is mainly carried out by using a hypha preservation method, and the hypha preservation method mainly comprises a distilled water preservation method, a liquid paraffin preservation method, a PDA (personal digital assistant) normal-temperature subculture preservation method, a PDA low-temperature preservation method, a liquid nitrogen preservation method and the like. The liquid nitrogen preservation method is mainly characterized in that wood rot fungi are inoculated on culture media with different matrixes, 5% -10% of glycerol is used as a protective agent, and the wood rot fungi are placed in a liquid nitrogen tank for long-term preservation after being cooled to-90 ℃ by a programmed cooling instrument.

Because the liquid nitrogen preservation method has wide application range, the survival rate after recovery is high and the biological activity can be kept, the method is widely adopted.

At present, the problems of low survival rate and low germination rate of strains after being frozen and stored are solved.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a method for freezing and preserving wood rotting fungi so as to solve the technical problem.

Aiming at the problems of low survival rate and low germination rate of the frozen strain after recovery, the inventor finds that the low survival rate of the frozen strain after recovery is caused by the fact that part of a culture medium with the strain to be preserved is placed in a freezing storage tube for storage in the existing freezing storage method, and taking a PDA culture medium as an example, because the PDA culture medium has high water content, mycelium cells are easy to expand and extrude due to ice crystals formed by water at low temperature in the process of program cooling or recovery and temperature return, the cells are damaged, the survival rate of the mycelium is reduced, and the germination rate is low.

The inventor provides a new idea to solve the technical problems and provides a new preservation substrate to ensure the survival rate of the strains after recovery.

The invention is realized by the following steps:

a freezing preservation method of wood rotting fungi comprises the following steps: and (3) taking the wood chip particles as carriers, and after the hypha of the wood rot fungi to be preserved spreads and grows to the interior of the wood chip particles, performing freeze preservation on the wood chip particles growing the wood rot fungi to be preserved.

Use the saw-dust granule as the carrier, inside wood rot fungus hypha grows to natural host saw-dust granule, the degradation saw-dust forms the porous structure who wraps up the mycelium, will have the saw-dust granule of wood rot fungus directly to carry out the refrigeration preservation, because saw-dust granule water content is low and wood rot fungus adapts to the saw-dust environment, both can avoid the destruction of the culture medium that the water content is high to the mycelium cell, can follow the quick activation in the natural host again to promote survival rate and the germination rate of freezing the back mycelium of preserving.

Compared with the conventional method, the thalli preserved by the freezing preservation method provided by the invention has the advantages that the germination speed is high, and the survival rate is greatly improved.

The method provided by the invention has the following beneficial effects: the survival rate of the mycelium is high, the germination speed is high, the mycelium grows vigorously, and the problems that wood rot fungi are easy to pollute, mutate and even die in the using and passage processes, so that the strains are declined and the strains are lost are solved.

In one embodiment, the cryopreservation method comprises: firstly, placing wood chip particles on a culture medium, then inoculating wood rot fungi to be preserved to the culture medium, culturing until hyphae spread and grow to the interior of the wood chip particles, and then freezing and preserving the wood chip particles growing the wood rot fungi to be preserved.

It is considered that hyphae have spread and grown inside the wood chip particles when the hyphae are observed to cover the entire wood chip particles with naked eyes and cultured for 3 to 5 days. In other embodiments, the determination may be made based on the growth time.

In one embodiment, the wood rot fungus to be preserved is cultured on the culture medium at a temperature of 15 to 30 ℃.

In one embodiment, the incubation time is 8-20 days.

In a specific embodiment, the culture temperature and the culture time are adaptively adjusted according to the type of wood rot fungi to be cultured, and are not limited to the temperature and the time defined in the present invention.

In one embodiment, the cryopreservation method comprises: inoculating wood rot fungi to be preserved to a culture medium for primary culture, then placing wood chip particles in the culture medium after the primary culture for secondary culture, and after hyphae of the wood rot fungi spread and grow to the interior of the wood chip particles, freezing and preserving the wood chip particles growing the wood rot fungi to be preserved. This embodiment requires special care to prevent excessive hyphal aging.

In one embodiment, the time for the first culture is 3-20 days, and the time for the second culture is 7-20 days.

In one embodiment, the temperature of the first culture is 15-30 ℃ and the temperature of the second culture is 15-30 ℃.

In other embodiments, the temperature and time of the primary culture and the secondary culture can be adaptively adjusted according to the need of culturing the strain, and are not limited to the temperature and time ranges given above in the present invention.

In one embodiment, the medium is a plate medium or a slant medium. The medium may be a plate or a slant medium, depending on the enrichment requirement.

In one embodiment, the culture medium is a PDA culture medium, a PSA culture medium, a potato integrated culture medium, a RM culture medium, a potato chip frying agar culture medium, a chip wheat bran agar culture medium, a rice bran frying agar culture medium, a wood rot fungi standard culture medium, a malt extract agar culture medium, or an agaricus culture medium.

The PDA culture medium is potato glucose agar culture medium, and can be prepared by itself or selected from commercially available PDA culture media.

The PSA culture medium is a potato sucrose agar culture medium and can be prepared by self.

The potato comprehensive culture medium comprises 200g of potato, 20g of glucose, 3g of potassium dihydrogen phosphate, 1.5g of magnesium sulfate, 15-10mg of vitamin B, 20g of agar and 1000ml of water.

The RM culture medium is a complete culture medium and can be prepared by self.

The potato sawdust decoction agar medium comprises 200g of potato, 20g of broadleaf tree sawdust (decoction), 20g of glucose, 20g of maltose, 20g of agar and 1000ml of water.

200g of potato culture medium with sawdust and wheat bran agar, 40g of broad-leaved tree wood sawdust (decoction), 20g of wheat bran (decoction), 20g of glucose, 20g of agar and 1000ml of water.

Rice bran decoction agar medium rice bran 40-100g (decoction), peptone 5g, magnesium sulfate 0.2g, potassium dihydrogen phosphate 0.3g, glucose 20g, agar 20-25g, and water 1000 ml.

The standard culture medium of wood rotting fungi comprises 20-25g of maltose extract, 10g of peptone, 20g of agar and 1000ml of water.

The malt extract agar culture medium comprises 20g of malt extract, 1 g of peptone, 20g of glucose, 20g of agar and 1000ml of water.

The agaricus bisporus culture medium comprises 10g of malt extract, 0.5 g of yeast extract, 0.5 g of magnesium sulfate, 0.5 g of calcium nitrate, 1.5g of peptone, 5g of maltose, 0.25 g of monopotassium phosphate, 20g of agar and 1000ml of water.

In addition, in other embodiments, other culture mediums capable of culturing wood rot fungi can be selected, and any culture medium capable of satisfying the growth of wood rot fungi is within the protection scope of the present invention.

In one embodiment, the wood chip particles are selected from wood chip particles of broad-leaved trees.

The wood particles of the broad-leaved trees can provide a substrate necessary for the growth of wood rotting fungi.

In one embodiment, the broad-leaved tree is at least one of oak, apple, camphor, birch, alstonia, yucca, magnolia, rose hip, cypress, weeping willow, elm, albizia julibrissin, robinia, Japanese pagodatree, populus davidiana, pomegranate, crape myrtle, prunus cerasifera, cottonrose hibiscus, french holly, cinnamomum japonicum, and dulcamara.

In another embodiment, the hardwood species may be selected as needed, and is not limited to the species of the hardwood species defined above in the present invention.

In one embodiment, the wood chip particles are aseptically processed wood chip particles; the aseptic processing step comprises the steps of pre-wetting the wood chip particles to be processed by water and then carrying out sterilization processing.

And (4) performing aseptic treatment to avoid the wood rotting fungi pollution caused by the mixed fungi carried by the wood dust particles. The wood chip particles are thoroughly sterilized by a pre-wetting treatment. In other embodiments, commercially available wood chip particles that have been sterilized may also be selected.

In one embodiment, the aseptic processing step comprises: fully soaking the wood chip particles to be treated in water, boiling for 20-30min, draining, and then performing steam sterilization at the temperature of 119-122 ℃. In other embodiments, the sterilization method may be selected as needed, and is not limited to the steam sterilization method described above.

In one embodiment, the wood chip particles are flake or block wood chip particles with a diameter of 3-8mm and a thickness of 2-5 mm.

The inventor finds out through practice that the wood chip particles with the particle size just meet the requirement of freely taking and placing the freezing storage pipe, in other embodiments, the size of the wood chip particles can be selected according to requirements, for example, the block-shaped wood chip particles with the diameter larger than 8mm or smaller than 3mm are also feasible, and the thickness larger than 5mm is also within the scope of the invention concept.

In one embodiment, the freeze preservation of the wood chip particles grown with wood rot fungi to be preserved comprises: placing the wood dust particles growing with the wood rot fungi to be preserved in a freezing storage tube, adding a freeze-drying protective agent into the freezing storage tube, then placing the freezing storage tube in a program cooling instrument for cooling, and placing the freezing storage tube at a low temperature for preservation after cooling.

It should be noted that the present invention provides only one specific freezing and temperature-reducing method, and in other specific practical processes, the temperature can be controlled for freezing and preservation as required, and is not limited to the method of temperature reduction in a program temperature-reducing instrument.

In one embodiment, the temperature reduction program of the programmed temperature reduction instrument is: at normal temperature, the temperature is reduced to 4 ℃ at a speed of 4-5 ℃/min, and then the temperature is reduced to-90 ℃ at a speed of 0.8-1.2 ℃/min.

The cooling program is only a cooling method provided by the invention, and in a specific practical process, the cooling program can also automatically select to increase the cooling rate or delay the cooling rate according to needs.

In one embodiment, the freezing tube is stored in a liquid nitrogen tank after the temperature is reduced. In other embodiments, storage in other low temperature environments may be selected, such as ultra-low temperature refrigerators.

In one embodiment, the cryopreservation method further comprises a strain recovery step, wherein the strain recovery step comprises the following steps: and taking the freezing tube out of the liquid nitrogen tank, carrying out constant-temperature water bath, and transferring to a culture medium to carry out recovery culture on the strains after the wood dust particles with wood rot fungi are completely melted.

In one embodiment, the constant temperature water bath is a water bath at 36-38 ℃; the constant temperature water bath time is 1-8 min. In other embodiments, the wood chip particles can be melted directly in hot water at a higher temperature or at normal temperature.

The wood rot fungi is selected from any one of the following fungi: lentinus edodes, oyster mushroom, needle mushroom, pleurotus eryngii, agaric, tremella, grifola frondosa, hericium erinaceus, hypsizigus marmoreus, agrocybe cylindracea, pleurotus geesteranus and the like.

In another embodiment, the wood rot fungi may be selected as needed, and the selection is not limited to the above-mentioned wood rot fungi. For example, other edible fungi may be used: poria, Boletus, Ganoderma, and Morchella.

The invention has the following beneficial effects:

the invention provides a cryopreservation method of wood rot fungi, which takes wood dust particles as a carrier, and the wood dust particles have a porous structure, so that hyphae of the wood rot fungi can spread and grow into the porous structure of the wood dust particles, and the wood dust particles with the wood rot fungi are directly subjected to cryopreservation, so that the damage of a culture medium with high water content to mycelium cells can be avoided, and the survival rate and the germination rate of the fungi are improved. The method provided by the invention has the following beneficial effects: the survival rate of the mycelium is high, the germination speed is high, the mycelium grows vigorously, and the problems that wood rot fungi are easy to pollute, mutate and even die in the using and passage processes, so that the strains are declined and the strains are lost are solved.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 shows the growth of Lentinus edodes strain Qihe No. 2 after being recovered by liquid nitrogen cryopreservation for 1 year with oak grains as matrix;

FIG. 2 shows the growth of Lentinus edodes strain L808 after recovery from cryopreservation with oak grains as matrix for 1 year in liquid nitrogen;

FIG. 3 shows the growth of Pleurotus ostreatus strain 3015 after 1 year resuscitation in liquid nitrogen cryopreservation with oak particles as the substrate;

FIG. 4 shows the growth of needle mushroom after low-temperature storage in liquid nitrogen for 1 year with oak grains as matrix;

FIG. 5 shows the growth of Pleurotus eryngii after low temperature storage for 1 year with oak grains as matrix and liquid nitrogen recovery;

FIG. 6 shows the growth of Grifola frondosa after being recovered by liquid nitrogen cryopreservation for 1 year using oak particles as matrix;

FIG. 7 shows the growth of Hericium erinaceus after low-temperature storage in liquid nitrogen for 1 year with oak grains as matrix;

FIG. 8 shows the growth of Lentinus Edodes strain QIHE No. 2 after recovery at low temperature with PDA as matrix and liquid nitrogen for 1 year;

FIG. 9 shows the growth of Lentinus edodes L808 after recovery from cryopreservation with PDA as matrix in liquid nitrogen for 1 year;

FIG. 10 shows the growth of Pleurotus ostreatus 3015 after recovery from cryopreservation in liquid nitrogen for 1 year using PDA as substrate;

FIG. 11 shows the growth of needle mushroom after being recovered by low temperature storage in 1 year with PDA as substrate and liquid nitrogen;

FIG. 12 shows the growth of Pleurotus eryngii after recovery at low temperature with PDA as substrate and liquid nitrogen for 1 year;

FIG. 13 shows the growth of Grifola frondosa after recovery from liquid nitrogen cryopreservation for 1 year using PDA as substrate;

FIG. 14 shows the growth of Hericium erinaceus after recovery in a low temperature storage with PDA as a substrate and liquid nitrogen for 1 year;

FIG. 15 shows the growth of Lentinus edodes strain before preservation in liquid nitrogen No. seven river 2;

FIG. 16 shows the growth of Pleurotus ostreatus 3015 before liquid nitrogen preservation;

FIG. 17 shows the growth of Pleurotus eryngii before liquid nitrogen preservation;

FIG. 18 shows the growth of Flammulina velutipes before liquid nitrogen preservation;

FIG. 19 shows the growth of Hericium erinaceus before liquid nitrogen preservation;

FIG. 20 shows the growth of Grifola frondosa before liquid nitrogen storage.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

This example provides a cryopreservation method for the Lentinus edodes strain No. Heqi river 2 and Lentinus edodes strain L808. The method comprises the following steps of:

taking oak particles as a preservation substrate, making the oak particles into a sheet or block sawdust particle substrate with the diameter of 3-8mm and the thickness of 2-5mm, and performing pre-wetting treatment and sterilization treatment; and under an aseptic condition, spreading the wood chip particle substrate on a PDA (personal digital assistant) plate to prepare a mixed culture medium of the wood chips and the PDA. (in this example, oak particles are used as the preservation substrate, and in other examples, other particles such as apple wood may be used as the preservation substrate).

Then, the mycelium to be preserved is inoculated on the mixed culture medium in a multi-point inoculation mode, and is placed in an incubator at 25 +/-1 ℃ for culturing for 15 days until the mycelium spreads and grows to the interior of the wood chip particles to form mycelium particles. (Lentinus Edodes strains Qihe No. 2 and Lentinus Edodes strains L808 separately cultured)

And then placing the mycelium particles (namely the sawdust particles with mycelium) into an aseptic freezing storage tube, injecting 5% of glycerol into the freezing storage tube to prepare a glycerol tubule, cooling by a programmed cooling instrument, and quickly placing the glycerol tubule into a liquid nitrogen tank for storage for 1 year. The cooling procedure is as follows: firstly, the temperature is reduced to 4 ℃ at the normal temperature of 25 ℃ at the speed of 5 ℃/min, and then the temperature is reduced to-90 ℃ at the speed of 1.2 ℃/min.

When the culture medium is recovered in the preservation period of 1 year, the freezing tube is quickly taken out of a liquid nitrogen tank, the wood chip hypha particles are completely melted by a constant-temperature water bath at 36 ℃ for 5min, and then transferred to a PDA culture medium and cultured at the temperature of 25 +/-1 ℃ until the hypha germinates.

The statistical result shows that the survival rate of the oak particles is more than 95 percent.

Example 2

This example provides a method for cryopreservation of Pleurotus ostreatus strain 3015. The method comprises the following steps of:

taking oak particles as a preservation substrate, making the oak particles into a sheet or block sawdust particle substrate with the diameter of 3-8mm and the thickness of 2-5mm, and performing pre-wetting treatment and sterilization treatment; and (3) under an aseptic condition, paving the sawdust particle substrate on a PDA (personal digital assistant) plate to prepare a mixed culture medium of sawdust and PDA. (in this example, oak particles are used as the preservation substrate, and in other examples, other particles such as apple wood may be used as the preservation substrate).

Then, the mycelium to be preserved is inoculated on the mixed culture medium in a multi-point inoculation mode, and is placed in an incubator at 25 +/-1 ℃ for culture for 14 days until the mycelium spreads and grows to the interior of the wood chip particles to form mycelium particles.

Placing the mycelium particles (i.e. sawdust particles with mycelium) in a sterile freezing storage tube, injecting 10% glycerol into the freezing storage tube to prepare a glycerol tubule, cooling by a programmed cooling instrument, and rapidly placing in a liquid nitrogen tank for storage for 1 year. The cooling procedure is as follows: firstly, the temperature is reduced to 4 ℃ at the normal temperature of 25 ℃ at the speed of 5 ℃/min, and then the temperature is reduced to-90 ℃ at the speed of 1.2 ℃/min.

Example 3

The embodiment provides a method for freezing and preserving flammulina velutipes. The method comprises the following steps of:

taking oak particles as a preservation substrate, making the oak particles into a sheet or block sawdust particle substrate with the diameter of 3-8mm and the thickness of 2-5mm, and performing pre-wetting treatment and sterilization treatment;

making a PDA plate, inoculating needle mushroom mycelia in a PDA culture medium at multiple points for primary culture for 3 days, and forming 0.5mm colonies after the mycelia are fixedly planted and germinate;

then, under aseptic conditions, the wood chip particle substrate is spread on a PDA plate (inoculated with flammulina velutipes hyphae for 3 days). (in this example, oak particles are used as the preservation substrate, and in other examples, other particles such as apple wood may be used as the preservation substrate). Culturing in a secondary incubator at 23 + -1 deg.C for 14d until the mycelium overgrows into the wood dust particles to form mycelium particles.

When the culture medium is recovered in the preservation period of 1 year, the freezing tube is quickly taken out of a liquid nitrogen tank, the wood chip hypha particles are completely melted by a constant-temperature water bath at 36 ℃ for 5min, and then transferred to a PDA culture medium and cultured at the temperature of 23 +/-1 ℃ until the hypha germinates.

Example 4

The embodiment provides a method for freezing and preserving pleurotus eryngii. The method comprises the following steps of:

Then, the mycelium to be preserved is inoculated on the mixed culture medium in a multi-point inoculation mode, and is placed in an incubator at the temperature of 23 +/-1 ℃ for culture for 14 days until the mycelium spreads and grows to the interior of the wood chip particles to form mycelium particles.

Example 5

The embodiment provides a freezing preservation method of grifola frondosa. The method comprises the following steps of:

Then, the mycelium to be preserved is inoculated on the mixed culture medium in a multi-point inoculation mode, and the mixed culture medium is placed in an incubator at 25 +/-1 ℃ for culturing for 20 days until the mycelium spreads and grows to the interior of the wood chip particles to form mycelium particles.

Example 6

The embodiment provides a cryopreservation method of hericium erinaceus. The method comprises the following steps of:

Example 7

Compared with the example 1, the method is only different in that apple wood particles are used as a substrate in the example, the shiitake strain No. Hejiang 2 and the shiitake strain L808 are respectively subjected to liquid nitrogen low-temperature preservation for 1 year, and the specific preservation method and the recovery method are the same as those in the example 1.

Example 8

Compared with example 2, the difference is that in this example, apple wood particles are used as a substrate, and the specific preservation method and the recovery method are the same as those in example 2.

Example 9

Compared with example 3, the difference of this example is only that apple wood particles are used as a substrate, a culture medium is a PSA culture medium, and the rest of the preservation method and the recovery method are the same as those of example 3.

Example 10

The present example is different from example 4 only in that elm particles are used as a substrate, the culture medium is sawdust and wheat bran agar medium, and the rest of the preservation method and the recovery method are the same as example 3. 200g of potato culture medium with sawdust and wheat bran agar, 40g of broad-leaved tree wood sawdust (decoction), 20g of wheat bran (decoction), 20g of glucose, 20g of agar and 1000ml of water. .

Comparative example 1

and (3) taking PDA as a preservation substrate, inoculating the mycelium to be preserved on the PDA plate according to a multi-point inoculation mode, and culturing in an incubator at 25 ℃ for 15 days until the mycelium grows to form mycelium particles. (Lentinus Edodes strains Qihe No. 2 and Lentinus Edodes strains L808 separately cultured)

And then placing the mycelium particles into a sterile freezing storage tube, injecting 5% of glycerol into the freezing storage tube to prepare a glycerol tubule, cooling by a programmed cooling instrument, and quickly placing the glycerol tubule into a liquid nitrogen tank for storage for 1 year. The cooling procedure is as follows: firstly, the temperature is reduced to 4 ℃ at the normal temperature of 25 ℃ at the speed of 5 ℃/min, and then the temperature is reduced to-90 ℃ at the speed of 1.2 ℃/min.

When the culture medium is recovered in the preservation period of 1 year, the freezing tube is quickly taken out from a liquid nitrogen tank, and is subjected to constant-temperature water bath at 36 ℃ for 5min, hypha particles are completely melted and then transferred to a PDA culture medium, and the PDA culture medium is placed at 25 ℃ for culture until hypha germinates.

Comparative example 2

and (3) taking PDA as a preservation substrate, inoculating the mycelium to be preserved on the PDA plate according to a multi-point inoculation mode, and culturing in an incubator at 25 +/-1 ℃ for 14 days until the mycelium grows to form mycelium particles.

And then placing the mycelium particles into a sterile freezing storage tube, injecting 10% of glycerol into the freezing storage tube to prepare a glycerol tubule, cooling by a programmed cooling instrument, and quickly placing the glycerol tubule into a liquid nitrogen tank for storage for 1 year. The cooling procedure is as follows: firstly, the temperature is reduced to 4 ℃ at the normal temperature of 25 ℃ at the speed of 5 ℃/min, and then the temperature is reduced to-90 ℃ at the speed of 1.2 ℃/min.

When the culture medium is recovered in the preservation period of 1 year, the freezing tube is quickly taken out from a liquid nitrogen tank, and is subjected to constant-temperature water bath at 36 ℃ for 5min, hypha particles are completely melted and then transferred to a PDA culture medium, and the PDA culture medium is placed at the temperature of 25 +/-1 ℃ for culture until hypha germinates.

Comparative example 3

and (3) taking PDA as a preservation substrate, inoculating the mycelium to be preserved on the PDA plate according to a multi-point inoculation mode, and culturing in an incubator at 23 +/-1 ℃ for 14 days until the mycelium grows to form mycelium particles.

When the culture medium is recovered in the preservation period of 1 year, the freezing tube is quickly taken out from a liquid nitrogen tank, and is subjected to constant-temperature water bath at 36 ℃ for 5min, hypha particles are completely melted and then transferred to a PDA culture medium, and the PDA culture medium is placed at the temperature of 23 +/-1 ℃ for culture until hypha germinates.

Comparative example 4

Comparative example 5

and (3) taking PDA as a preservation substrate, inoculating the mycelium to be preserved on the PDA plate according to a multi-point inoculation mode, and culturing in an incubator at 25 +/-1 ℃ for 20 days until the mycelium grows to form mycelium particles.

Comparative example 6

Experimental example 1

The average survival rates of the mycelia after the resuscitations in examples 1 to 6 and comparative examples 1 to 6 were measured, and the average survival rates of the mycelia are shown in table 1, and it is understood from table 1 that the survival rate of the mycelia after the strain preservation using oak particles as the substrate was higher than that of the mycelia after the PDA substrate preservation.

FIG. 1 shows the growth of Lentinus edodes strain Qihe No. 2 after recovery in liquid nitrogen cryopreservation for 1 year with oak grains as matrix; FIG. 2 shows the growth of Lentinus edodes strain L808 after recovery from cryopreservation with oak grains as matrix for 1 year in liquid nitrogen; FIG. 3 shows the growth of Pleurotus ostreatus strain 3015 after 1 year resuscitation in liquid nitrogen cryopreservation with oak particles as the substrate; FIG. 4 shows the growth of needle mushroom after low-temperature storage in liquid nitrogen for 1 year with oak grains as matrix; FIG. 5 shows the growth of Pleurotus eryngii after low temperature storage for 1 year with oak grains as matrix and liquid nitrogen recovery; FIG. 6 shows the growth of Grifola frondosa after being recovered by liquid nitrogen cryopreservation for 1 year using oak particles as matrix; FIG. 7 shows the growth of Hericium erinaceus after being recovered by cryopreservation in liquid nitrogen for 1 year using oak grains as a matrix.

FIG. 8 shows the growth of Lentinus Edodes strain QIHE No. 2 after recovery at low temperature with PDA as matrix and liquid nitrogen for 1 year; FIG. 9 shows the growth of Lentinus edodes L808 after recovery from cryopreservation with PDA as matrix in liquid nitrogen for 1 year; FIG. 10 shows the growth of Pleurotus ostreatus 3015 after recovery from cryopreservation in liquid nitrogen for 1 year using PDA as substrate; FIG. 11 shows the growth of needle mushroom after being recovered by low temperature storage in 1 year with PDA as substrate and liquid nitrogen; FIG. 12 shows the growth of Pleurotus eryngii after recovery at low temperature with PDA as substrate and liquid nitrogen for 1 year; FIG. 13 shows the growth of Grifola frondosa after recovery from liquid nitrogen cryopreservation for 1 year using PDA as substrate; FIG. 14 shows the growth of Hericium erinaceus after recovery from cryopreservation in liquid nitrogen for 1 year using PDA as a substrate.

FIG. 15 shows the growth of Lentinus edodes strain before preservation in liquid nitrogen No. seven river 2; FIG. 16 shows the growth of Pleurotus ostreatus 3015 before liquid nitrogen preservation; FIG. 17 shows the growth of Pleurotus eryngii before liquid nitrogen preservation; FIG. 18 shows the growth of Flammulina velutipes before liquid nitrogen preservation; FIG. 19 shows the growth of Hericium erinaceus before liquid nitrogen preservation; FIG. 20 shows the growth of Grifola frondosa before liquid nitrogen storage.

Comparing fig. 1 and fig. 8, it can be seen that the strain growth is better when the oak particles provided by the present invention are used as the substrate and the liquid nitrogen preservation method is adopted than when the PDA substrate preservation method of comparative example 1 is adopted under the same recovery time.

Similarly, the same conclusion can be drawn by comparing fig. 2 and 9.

Table 1 hypha average survival statistical table.

Experimental example 2

In the experimental example, feasibility experiments are carried out on different wood rot fungus strains by using oak with different granularity as a liquid nitrogen preservation matrix.

Three oak sawdust with different granularities of 3-5mm, 5-6mm and 6-8mm are respectively designed as preservation matrixes for liquid nitrogen preservation of wood rot fungus strains, liquid nitrogen preservation is respectively carried out on champignon No. 2, champignon L808, oyster mushroom, pleurotus eryngii, flammulina velutipes, grifola frondosa and hericium erinaceus, and the hypha germination rate is revived and observed after 1 year.

Specifically, according to the preservation method of example 1, sheet-like or block-like oak chip particle substrates with particle sizes of 3-5mm, 5-6mm and 6-8mm are subjected to pre-wetting treatment and sterilization treatment; under the aseptic condition, spreading oak sawdust particle matrixes with different granularities on a PDA (personal digital assistant) plate respectively to prepare a mixed culture medium of sawdust and PDA; inoculating mycelia to be preserved on a mixed culture medium/PDA (potato dextrose agar) according to a multi-point inoculation mode, and culturing in an incubator at 25 ℃ for 14 days until the mycelia overgrow and grow into wood chip particles/PDA matrix to form mycelia particles; then placing the mycelium particles into a sterile freezing storage tube, injecting 10% glycerol into the freezing storage tube to prepare a glycerol tubule, cooling by a programmed cooling instrument, and quickly placing the glycerol tubule into a liquid nitrogen tank for storage for 1 year.

Storing in liquid nitrogen tank for 1 year, taking out the freezing tube from the liquid nitrogen tank, thawing in 36-38 deg.C water bath for 3min, transferring wood chip hypha particles to PDA culture medium after completely melting, and culturing at 25 + -1 deg.C until hypha germinates. And finally, counting the survival rate of hyphae. As shown in Table 2 below, it can be seen from Table 2 that the size of oak grains has no significant effect on the survival rate of the strain.

Table 2 results of feasibility experiments were performed on quercus robur of different particle sizes as a liquid nitrogen preservation substrate.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A freezing preservation method of wood rotting fungi is characterized by comprising the following steps: and (3) taking the wood chip particles as carriers, and after the hypha of the wood rot fungi to be preserved spreads and grows to the interior of the wood chip particles, performing freeze preservation on the wood chip particles growing the wood rot fungi to be preserved.

2. The cryopreservation method of wood rot fungi according to claim 1, wherein the cryopreservation method comprises: firstly, placing wood chip particles on a culture medium, then inoculating wood rot fungi to be preserved to the culture medium, culturing until hyphae spread and grow to the interior of the wood chip particles, and then freezing and preserving the wood chip particles growing the wood rot fungi to be preserved.

3. The cryopreservation method of wood rot fungi according to claim 2, wherein the culture temperature of the wood rot fungi to be preserved on the culture medium is 15 to 30 ℃;

preferably, the culture time is 8-20 d.

4. The cryopreservation method of wood rot fungi according to claim 1, wherein the cryopreservation method comprises: inoculating wood rot fungi to be preserved to a culture medium for primary culture, then placing wood chip particles in the culture medium after the primary culture for secondary culture, and after hyphae of the wood rot fungi grow to the interior of the wood chip particles in a spreading manner, performing freeze preservation on the wood chip particles growing the wood rot fungi to be preserved.

5. The cryopreservation method of wood rot fungi according to claim 4, wherein the time for the primary culture is 3-20d, and the time for the secondary culture is 7-20 d;

preferably, the temperature of the primary culture is 15-30 ℃, and the temperature of the secondary culture is 15-30 ℃.

6. The cryopreservation method of wood rot fungi according to any one of claims 2 to 5, wherein the culture medium is a plate culture medium or a slant culture medium;

preferably, the culture medium is a PDA culture medium, a PSA culture medium, a potato comprehensive culture medium, an RM culture medium, a potato sawdust frying juice agar culture medium, a sawdust wheat bran agar culture medium, a rice bran frying juice agar culture medium, a wood rot fungi standard culture medium, a malt extract agar culture medium or an agaricus bisporus culture medium.

7. The method for cryopreservation of wood rot fungi according to any one of claims 1 to 6, wherein the wood chip particles are selected from wood chip particles of broad-leaved trees;

preferably, the broad-leaved tree is at least one of oak, cyclobalanopsis glauca, chestnut, birch, alder, tannin extract, apple, camphor, silver birch, alstonia, white yucca, magnolia flower, red peach, cypress, weeping willow, elm, albizia julibrissin, acacia, Japanese pagodatree, populus davidiana, pomegranate, crape myrtle, red plum, cotton rose, French holly, cinnamomum japonicum, sweetgum and dulcis;

preferably, the wood chip particles are aseptically processed wood chip particles; the aseptic processing step comprises the steps of prewetting the wood chip particles to be processed with water and then carrying out sterilization processing;

preferably, the aseptic processing step comprises: fully soaking the wood dust particles to be treated in water, boiling for 20-30min, draining, and then performing steam sterilization at the temperature of 119-;

preferably, the wood chip particles are flake or block wood chip particles with the diameter of 3-8mm and the thickness of 2-5 mm.

8. The cryopreservation method of wood rot fungi according to claim 1, wherein the cryopreservation of the wood chip particles grown with wood rot fungi to be preserved comprises: placing the wood chip particles growing with wood rot fungi to be preserved in a freezing storage tube, adding a freezing protective agent into the freezing storage tube, then placing the freezing storage tube in a program cooling instrument for cooling, and preserving the freezing storage tube at a low temperature after cooling;

preferably, the temperature reduction program of the program temperature reduction instrument is set as follows: reducing the temperature to 4 ℃ at normal temperature at a speed of 4-5 ℃/min, and then reducing the temperature to-90 ℃ at a speed of 0.8-2 ℃/min;

preferably, the cryoprotectant is: 5-10% glycerol;

preferably, the freezing tube is stored in a liquid nitrogen tank after being cooled.

9. The cryopreservation method of wood rot fungi according to claim 8, further comprising the step of recovering the strain, wherein the recovering of the strain comprises the following steps: taking the freezing tube out of the liquid nitrogen tank, carrying out constant-temperature water bath, and transferring to a culture medium for recovery culture of strains after wood dust particles with wood rot fungi are completely melted;

preferably, the thermostatic water bath is a water bath at 30-38 ℃; the constant temperature water bath time is 1-8 min.

10. The cryopreservation method of wood rot fungi according to claim 1, wherein the wood rot fungi is selected from any one of the following fungi: lentinus edodes, oyster mushroom, needle mushroom, Pleurotus eryngii, Auricularia, Tremella, Grifola frondosa, Hericium erinaceus, Hypsizygus marmoreus, Agrocybe cylindracea and Pleurotus geesteranus.